Mitosis is one of the most spectacular events in cell biology, and over the past two decades much progress has been made towards understanding its regulation. Here we propose four Specific Aims centered on key regulators of the mitotic kinase Cdk1. (1) To identify which cyclin(s) is/are required for mitotic entry in HeLa cells. With the advent of effective RNAi methods, we can now ask which cyclins are required for M-phase entry. Somewhat surprisingly, we have found that cyclin A2 is the most important single cyclin for the G2/M transition. We now plan to determine whether A2 is required to regulate the activation or localization of B1/B2, or whether it plays a more direct role in mediating M-phase entry. We also hope to unambiguously determine the relevant Cdk partner for A2's mitotic function through rescue experiments with Cdk-cyclin fusion proteins. (2) To elucidate the mechanism of Wee1 inactivation. We plan to dissect the interaction of Cdk1 -cyclin B1 with Wee1A and to determine the mechanism that generates Wee1A's markedly ultrasensitive response. We also plan to identify the kinases that regulate Wee1A. (3) To elucidate the mechanism of Cdc25C regulation. We have found that the N-terminus of Cdc25C functions as an inhibitor of Cdc25C activity, and that purified N-terminus can inhibit the catalytic domain in trans. Our working hypothesis is that phosphorylation prevents the N-terminus of Cdc25C from inhibiting the catalytic domain. We plan to distinguish among different possible mechanisms for this inhibition through a combination of in vitro kinetic studies and structural studies. (4) To determine what triggers mitosis in HeLa cells. In HeLa cells, some unidentified regulator or regulators determines the timing of cyclin B1-Cdk1 activation and nuclear translocation. We plan to carry out a systematic RNAi screen to identify these regulators.